鼻疽诺卡菌NFA47650蛋白免疫保护作用评价

  目的  评估鼻疽诺卡菌NFA47650蛋白激活固有免疫信号通路的能力，并评价其免疫保护效果。  方法  利用生物信息学分析nfa47650序列特征，Blast比对其在鼻疽诺卡菌中的保守性；用纯化后的NFA47650蛋白刺激RAW264.7细胞，利用Western Blot免疫印迹检测MAPK信号通路激活水平、利用ELISA检测细胞上清中细胞因子诱导水平；将C57BL/6小鼠分成两组，分别免疫PBS及NFA47650蛋白3次，检测血清效价后感染鼻疽诺卡菌，并检测感染24 h后小鼠体温、体重变化、血清IgG水平、肺泡灌洗液中蛋白水平、LDH水平以及肺部菌载量、肺上清细胞因子等指标。  结果  NFA47650蛋白能够刺激RAW264.7细胞上调ERK、JNK、P38磷酸化水平，并诱导IL-10、IL-12、TNF-α、IFN-γ等多种细胞因子浓度升高。 另外，该蛋白免疫小鼠后可以产生高效价的抗血清，使肺部感染减轻，肺部菌载量减少。   结论  NFA47650蛋白作为一种免疫显性蛋白，能在鼻疽诺卡菌感染宿主过程中刺激机体产生抗体，诱导小鼠产生免疫应答，具有免疫保护作用。

Evaluation of immunoprotective effect of NFA47650 protein from Nocardia farcinica

  Objective  To access the capacity of NFA47650 protein from Nocardia farcinica in activating innate immune response and evaluate its immunoprotective effect.   Methods  The sequence characteristics of nfa47650 were analyzed by bioinformatics, and its conservation in N. farcinica was analyzed by Blast. RAW264.7 cells were stimulated with purified NFA47650 protein, the level of MAPK signaling pathway activation was detected by Western Blot, and the concentration of cytokines in the supernatant were measured by ELISA. C57BL/6 mice were divided into two groups to be immunized with PBS or NFA47650 protein for three times, respectively. After serum titer was detected, the changes of body temperature, body weight, serum IgG level, protein and LDH level in alveolar lavage fluid, pulmonary bacterial loads and cytokine levels were detected at 24 hours after infection.   Results  NFA47650 protein can stimulate RAW264.7 cells to upregulate the phosphorylation levels of ERK, JNK, P38 and induces the production of various cytokines such as IL-10, IL-12, TNF-α, IFN-γ. In addition, immunized mice produced high-titer antisera, resulting in less damage and bacterial loads in lung tissues.   Conclusion  NFA47650 protein is an immunodominant protein with immunoprotective effects that stimulates the host to produce antibodies and induces an immune response in mice during N. farcinica infection.